An optimised reverse transcriptase system for the production of full-length cDNA.
- Produce full-length cDNA with RNAse H- mutant
- Achieve higher specificity at elevated temperatures, up to 55°C
EpiScript™ Rnase H- Reverse Transcriptase (EpiScript RT), an alternative to SuperScript® II Reverse Transcriptase, is a recombinant MMLV reverse transcriptase with greatly reduced RNase H activity. It is highly efficient at producing full-length cDNA from long RNA templates. EpiScript RT is capable of producing cDNA from as little as 50 pg of total RNA for real-time RT-PCR (qRT-PCR) analysis and other applications.
- First-strand cDNA synthesis for subsequent PCR or real-time PCR.
- Recombinant MMLV reverse transcriptase with greatly reduced RNase H activity
- Active at temperatures up to 55°C
- Highly efficient at producing full-length cDNA from as little as 50 pg of total RNA
- Best value in an RNase H- Reverse Transcriptase
Storage: Store only at -20°C in a freezer without a defrost cycle.
Storage Buffer: EpiScript RT is supplied in a 50% glycerol solution containing 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 1 mM DTT, 0.1 mM EDTA, and 0.1% Triton® X-100.
Unit Definition: One unit of EpiScript RT catalyzes the incorporation of 1 nmol of dTTP into acid-insoluble material in 10 minutes at 37°C using saturating amounts of oligo(dT)-primed poly(rA) as template.
Contaminating Activity Assays: EpiScript RT is free of detectable exonuclease, endonuclease, and RNase activities.
Figure 1. EpiScript™ Reverse Transcriptase performed equally or better than comparable reverse transcriptases from other vendors. First- strand synthesis reactions were assembled according to manufacturer´s specifications. Input RNA was 1 µg of Jurkat total RNA (Ambion®). Reactions were primed using 50 ng of poly-T(16-18) DNA. 2nd strand qPCR was performed using Bio-Rad iQ SYBR mastermix and gene-specific primers that yielded 250-350 bp amplicons. Reactions were repeated 4-fold. PGDF-R (Platelet Derived Growth Factor Receptor), TNF (Tumor Necrosis Factor), IL-1b (Interleukin-1 beta), IL-2 (Interleukin 2). Image courtesy of Matthew Kellinger, Illumina® Inc.
|Figure 3. Use of EpiScript® Reverse Transcriptase for first strand cDNA results in detection of similar transcript categories independent of input amount. EpiScript RT was used to random prime 5 ng or 50 pg of human total RNA and the cDNA was converted into libraries for Illumina® sequencing. Reads were aligned using Tophat and annotated with Cufflinks and the percentage of each major category is visualised as a pie chart. Equivalent results are observed for both 5 ng and 50 pg of input RNA.|